EP0477972B1 - Séquences nucléotidiques utiles comme sondes spécifiques du type amorces de PCR et sondes pour l'amplification et détection du virus-papilloma humain, et kits et procédés utilisés dans ce but - Google Patents

Séquences nucléotidiques utiles comme sondes spécifiques du type amorces de PCR et sondes pour l'amplification et détection du virus-papilloma humain, et kits et procédés utilisés dans ce but Download PDF

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EP0477972B1
EP0477972B1 EP91116561A EP91116561A EP0477972B1 EP 0477972 B1 EP0477972 B1 EP 0477972B1 EP 91116561 A EP91116561 A EP 91116561A EP 91116561 A EP91116561 A EP 91116561A EP 0477972 B1 EP0477972 B1 EP 0477972B1
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papilloma virus
dna
human papilloma
test sample
oligonucleotide
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EP0477972A3 (en
EP0477972A2 (fr
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Jeffrey L. Joseph
Stanley R. Bouma
Ronald L. Marshall
Thomas G. Laffler
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Abbott Laboratories
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Definitions

  • This invention relates generally to human papilloma virus, and more particularly, relates to nucleotide sequences of short strands of human papilloma virus which can be amplified and/or used to determine the presence of human papilloma virus products in a test sample, and some of which also can be amplified and/or used to determine the specific type of human papilloma virus of types 16 and 18 present in the test sample.
  • HPV Human papilloma virus
  • HPV types namely, HPV type 16 and type 18, and to a lesser extent HPV types 31, 33 and 35, are found in a high proportion of invasive cervical cancers and their metastases.
  • HPV types which infect the anogenital tract such as HPV types 6 and 11
  • HPV types 31 , 33 and 35 HPV types which infect the anogenital tract
  • HPV types 16 and 18 HPV types 16 and 18
  • the molecular hybridization techniques used to detect homologous DNA sequences are sensitive and can be highly specific if used with probes which bind to nucleic acid sequences which are unique to a particular HPV type.
  • concentration of total viral DNA in a given clinical sample may be below the limit of sensitivity of the test. For example, the amount of viral DNA in dysplastic cervical lesions is reduced with increasing dysplasia.
  • viral DNA sequences can be amplified by using, for example, the polymerase chain reaction (PCR) or the ligase chain reaction (LCR) techniques.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the products thus obtained can be identified by using conventional hybridization techniques for identification of virus types, such as Southern blotting. See C. Oste, Biotechniques 6:163( 1988), K. B. Mullis, U. S. Patent No. 4,683,202, and EP-A-320 308 (BioTechnica).
  • Both PCR and LCR serve to amplify the DNA present in a test sample to detectable levels.
  • the level of sensitivity is about 50 to 100 copies per sample.
  • the next most sensitive technique is dot-blot, which can detect about 10,000 molecules, while Southern blot reliably detects about 100,000 copies of DNA per sample.
  • the appropriate diagnosis of HPV may require two steps.
  • the presence of a clinically relevant type of HPV is first detected with a group-specific primer.
  • differentiation between types can be performed by using a type-specific probe having low homology between the HPVs of the group.
  • differentiation can be performed using a mixture of type-specific probes at the outset, provided these probes amplify DNA independently of each other, and that they can be detected independently.
  • such tasks were attempted using specific antibodies.
  • nucleic acid probes and primers allow greater discrimination among subtypes than do antibodies. The use of DNA-based tests increases both sensitivity and specificity over prior-art antibody-based tests.
  • oligonucleotide strands of DNA which could be amplified and used to detect the presence, if any, of HPV in a test sample. It also would be advantageous to provide short oligonucleotide strands of DNA which could be amplified and used to detect the presence, if any, of specific types of HPV in the test sample.
  • the combined use of oligonucleotide strands would be advantgeous for allowing for the specific and sensitive in vitro diagnosis of the presence and specific type of HPV present in test samples.
  • Oligonucleotides of from about 10 to about 60 nucleotides which can be amplified and used either to detect specific sequences of specific types of human papilloma virus, or consensus regions with high homology among different types.
  • the presence of HPV is determined by contacting the test sample with sequences provided to detect the presence, if any, of HPV types 6, 11, 16, 18, 31, 33 and 61. This may be done with or without prior amplification, for example, by PCR or LCR. Either type-specific or consensus amplification is also possible.
  • Two oligonucleotides are provided if the sequence is to be amplified by PCR, and four oligonucleotides provided if amplification is by LCR, in accordance with these known amplification procedures.
  • the type of HPV present in the sample can be determined by using HPV type-specific probes, by subsequent rounds of PCR, or by LCR.
  • the presence of type-specific HPV can be determined by contacting the test sample directly with type-specific nucleotide sequence provided by the invention for the detection of HPV types 16 and 18.
  • FIG. 1 is a photograph of a gel following electrophoresis showing the results when the primers PCR 1 and PCR5 were used to amplify selected plasmids wherein HPV 6 is in lane 1, HPV 11 is in lane 2, HPV 16 is in lane 3, HPV 18 is in lane 4, and HPV 31 is in lane 5, HPV 33 is in lane 6, HPV 61 is in lane 7, and molecular weight standards are in lane 8.
  • FIG. 2 is a photograph of a gel following electrophoresis showing the results when the primers PCR 1, PCR2, PCR3, PCR4 and PCR5 were used to amplify plasmid p65.16.8 (HPV 16).
  • PCR1 and PCR5 are primers according to the invention.
  • FIG. 3 is a photograph of the ethidium bromide-stained gels wherein PCR 1 4 and PCR15 are used in conjunction with IWDO to obtain amplified PCR product.
  • FIG. 4 is a graph of results obtained from performing LCR on 10 7 molecules of the selected target using LCR5A, LCR5A', LCR5B and LCR5B'.
  • the rate of reaction of 4-methyl lumbelliferone is expressed as fluorescence counts/second/second and plotted against the target HPV type.
  • FIG. 5 is a graph of results obtained from performing LCR on 10 7 molecules of the selected target using LCR6A, LCR6A', LCR6B and LCR6B'.
  • the rate of reaction of 4-methyllumbelliferone is expressed as fluorescence counts/second/second and plotted against the target HPV type.
  • FIG. 6 is a graph of results obtained from perfomring LCR on 10 7 molecules of the selected target using LCR7A, LCR7A', LCR7B and LCR7B'.
  • the rate of reaction of 4-methyllumbelliferone is expressed as fluorescence counts/second/second and plotted against the target HPV type.
  • FIG. 7 is a graph of results obtained from perfomring LCR on 10 7 molecules of the selected target using LCR8A, LCR8A', LCR8B and LCR8B'.
  • the rate of reaction of 4-methyllumbelliferone is expressed as fluorescence counts/second/second and plotted against the target HPV type.
  • HPV HPV Advanced human immunodeficiency virus
  • the appropriate diagnosis of HPV requires two sets of conditions. The first enables the detection of all pertinent types, and the second set allows differentiation among them. In the past, such tasks have been attempted using specific antibodies. In general, nucleic acid probes and primers allow greater discrimination among subtypes than do antibodies. Thus, the use of DNA-based tests tends to increase both sensitivity and specificity over antibody-based tests.
  • two complementary polynucleotide strands are amplified by treating the strands with two oligonucleotide primers such that an extension product of each primer is synthesized which is complementary to each nucleic acid strand.
  • the primers are selected such that the extension product of one primer forms a template for the synthesis of an extension product from the other primer once the extension product of the one primer is separated from the template.
  • a chain reaction is maintained by a cycle of denaturing the primer extension products from their templates, treating the single-stranded molecule generated with the same primers to re-anneal, and allowing the primers to form further extension products. The cycle is repeated for any many times as it takes to increase the target nucleic acid segments to a concentration where they can be detected.
  • the amplified target sequence can be detected by any of several known techniques; for example, by denaturing the double-stranded products formed by PCR, and treating those products with one or more reporter probes which hybridize with the extension products.
  • the reporter probe has a detectable label, and usually is added in excess. The unhybridized reporter probe, therefore, must be separated from the hybridized reporter probe by involving a separation step.
  • the extension products are detected by gels stained with ethidium bromide. The diagnosis can be confirmed by transferring the DNA to nitrocellulose and probing with a probe specific to the HPV type suspected of being present in the sample.
  • hapten a different reporter molecule, e.g. hapten
  • hapten1 is attached to each primer.
  • duplexes can be detected by "capturing" one hapten (hapten1) with a solid phase coated with anti-hapten1.
  • the separated complex can be detected with a conjugate of label and anti-hapten2, and label associated with the solid phase can be measured.
  • the Ligase Chain Reaction amplifies sections of DNA by copying the section of DNA, and copying the copies of that section of DNA, many times over.
  • This method is described in European Patent Application No. 0 320 308 published June 14, 1989, which is incorporated herein by reference.
  • two probes for example, A and B
  • This ligated probe then is denatured away from the target, after which it is hybridized with two additional probes (A' and B') of sense opposite to the initial probes A and B.
  • the secondary probes are themselves then ligated. Subsequent cycles of denaturation/hybridization/ligation create the formation of double-length probes of both sense (+) and antisense (-).
  • the nucleic acid of the sample is provided either as single stranded DNA or as double-stranded DNA which is denatured to separate the strands.
  • Four probes are utilized: the first two probes (A and B) are the so-called primary probes, and the second two probes (A' and B') are the so-called secondary probes.
  • the first probe (A) is a single strand capable of hybridizing to a first segment of the primary strand of the target nucleotide sequence.
  • the second probe (b) is capable of hybridizing to a second segment of the primary strand of the target nucleotide sequence.
  • the 5' end of the first segment of the primary strand of the target is positioned relative to the 3' end of the second segment of the primary strand of the target to enable joining of the 3' end of the first probe to the 5' end of the second probe, when the probes are hybridized to the primary strand of the target nucleotide sequence.
  • the third probe (A') is capable of hybridizing to the first probe
  • the fourth probe (B') is capable of hybridizing to the second probe (B).
  • the hybridized probes are ligated to form reorganized fused probe sequences. Then, the DNA in the sample is denatured to separate ligated probes from sample DNA.
  • Successive cycles wherein the ligated probes and target DNA undergo the above-described process are performed to increase the amount of detectable DNA in the sample.
  • the amount of cycles performed is dependent upon the sequence used and the sensitivity required of the test. Usually, the cycle can be repeated from 15 to 60 times. At least one of the probes can be conjugated to a signal generating compound.
  • the detection of amplified product can be accomplished using standard manual or automated immunoassay procedures known to those skilled in the art. These procedures include, for example, immunochromatography, ELISA, EIA and MEIA. Hybridization also can be accomplished by following standard dot-, slot- or replica-blot procedures which are known to those in the art.
  • the sequences can be labelled with an appropriate signal generating compound (label), which is capable of generating a measureable signal detectable by external means.
  • the various signal generating compounds contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluoroscein and rhodamine, chemiluminescent compounds, radioactive elements such as 32 P, and other labels known to those of ordinary skill in the art.
  • the selection of a particular label is not critical, but it will be capable of producing a a signal either by itself or in conjunction with one or more additional substances.
  • a variety of different indicator reagents can be formed of label and specific binding member. Either the label or a specific binding member can be varied.
  • Examples of specific binding members which can be used as a member of the indicator reagent include antibodies, both monoclonal, polyclonal, and fragments thereof; avidin or biotin, biotin and anti-biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor or an enzyme; an enzyme inhibitor or an enzyme; also any antigenic substances, haptens, antibodies, and combinations thereof.
  • the test sample can be any biological material suspected of containing HPV.
  • the test sample can be human body tissue, or a test sample which contains cells suspected of containing HPV.
  • PCR was performed essentially following the package insert of the commercially available Gene-AmpTM kit (document No. 55635-6/89, available from Perkin-Elmer/Cetus, Emeryville, CA).
  • DNA polymerase Thermus Acquaticus 25 or 63.9 units/1 mL
  • reaction mixture was overlayed with 100 ⁇ L of mineral oil.
  • the tube then was placed in an instrument capable of incubation at several temperatures, and subjected to 30 or 40 cycles of programmed temperature change. The precise cycle of temperature change used, and the instrument used, varied with the experiment, and is detailed in the descriptions of the figures in Example 3.
  • Sequence IWDO is derived from a sequence disclosed in International application number PCT/US86/00629 (WO 86/05816).
  • TABLE 1 shows the sequences and where they map to to in the various types.
  • Linearized plasmids containing full-length papilloma virus inserts in pGEM3 were used as targets. These were pHPV6.1 (HPV6), pSP65.11.5 ( HPV 11), p65.16,8 (HPV16), pHPV18H(HPV18), pG3 HPV31 ( HPV31),pLNK322,HPV33 ( HPV33), and pBR322.HPV61 (HPV61).
  • the Programmable Cyclic ReactorTM available from Ericomp, San Diego was used as the incubation instrument.
  • FIG. 1 is a photograph of an ethidium bromide-stained 1.2% agarose gel showing results using 63.9 units/mL DNA polymerase, in the DNA Thermal CyclerTM (Perkin-Elmer/CETUS, Emeryville, CA). The samples were heated for 5 minutes at 94°C, then subjected to 40 cycles of a temperature program of: 1 minute at 94°C, 2 minutes at 40°C, and 1.5 minutes at 72°C. The PCR primers used in this case were PCR 1 and PCR5 of Example 2. Examination of the gel following electrophoresis showed bands at the expected positions, i.e. 292 bp.
  • Lane 1 HPV6; lane 2, HPV 11; lane 3, HPV16 ; lane 4, HPV 18; lane 5, HPV31; lane 6, HPV33, lane 7, HPV61; lane 8, pooled human placental DNA ( suspected of having HPV infection); lane 9, molecular weight markers-Hae III digest of ⁇ X174.
  • FIG. 2 is a photograph of an ethidium bromide-stained 4% agarose gel showing results using 25 units/mL DNA polymerase, in the Programmable Cycler ReactorTM ( Ericomp, San Diego, CA). Samples in this case were subjected to 30 cycles of a temperature program of: 50°C for one (1) minute, 72°C for two (2) minutes and 95°C for one (1) ) minute. In this case, the primers PCR1 , PCR2, PCR3, PCR4 and PCR5 of Example 2 were used to amplify plasmid p65,16,8(HPV 16).
  • FIG. 3 is a photograph of an ethidium bromide-stained 1.2% agarose gel showing results using the same conditions as FIG. 1.
  • PCR14 and PCR15 were used as primers in conjunction with IWDO.
  • the expected size of the amplified PCR product of PCR 14 and IWDO is 437 bp for all of the HPV types tested.
  • the expected size of the product of PCR 15 and IWDO is 98 bp. Products of these sizes appear in the gels, confirming that PCR14 and PCR15, used in conjunction with IWDO, will amplify HPV DNA of types 6, 11, 16, 18, 31 , 33, and 61.
  • Lane 1 Molecular weight marker (Hae III digest of FX 174); PCR 14 + IWDO, lanes 2-9: lane 2, HPV6 ; lane 3, HPV 11 ; lane 4, HPV16 ; lane 5, HPV18 ; lane 6, HPV31; lane 7, HPV33; lane 8, HPV61 ; lane 9, human placental DNA suspected of being infected with HPV; PCR 5 + IWDO, lanes 10-17: lane 10, HPV6 ; lone 11 , HPV 11; lane 12, HPV16; lane 13, HPV18 ; lone 14, HPV31 ; lane 15, HPV33 ; lane 16, HPV61 ; lane 17, human placental DNA suspected of being infected with HPV; lane 18, molecular weight marker ( Hae III digest of FX174 and HinD III digest of 1 DNA) .
  • This reaction mixture was overlayed with 30 ⁇ L of mineral oil.
  • the tube was placed in an instrument capable of incubation at several temperatures (e.g. thermal cycler from Coy Laboratory Products (Ann Arbor, MI) or the Programmable Cycler ReactorTM (available from Ericomp, San Diego, CA), and then subjected to several cycles of programmed temperature change. Each cycle involved incubation at 50°C for one minute and 85°C for one minute.
  • Example 4 The following procedure was used when performing the Ligase Chain Reaction ( LCR), which is described in published European Patent Application No. 0 320 308 A2.
  • the reagents of Example 4 were utilized in the procedure as follows: Two probes (A and B) complementary to immediately adjacent to regions of a target sequence were hybridized and ligated. This ligated probe was denatured away from the target, and hybridized with two additional probes (A' and B') of sense opposite to the initial probes (A and B). The secondary probes then were ligated. Subsequent cycles of denaturation/hybridization/ligation created the formation of double-length probes of both + and - sense.
  • LCR Ligase Chain Reaction
  • Base-denatured plasmids which contained full-length papilloma virus inserts in pGEM3 were used as targets. These plasm ids were pG3HPV6(+) (HPV6), pSP 65. 11.5 (HPV11), pSP65.168 (HPV16), p63HPV18H(-)(HPV18), p63:HPV31 (HPV31), pLNK322:HPV33 (HPV33), pBR322:HPV35 (HPV35), pUC19:HPV52 (HPV52), pLNK322:HPV58 (HPV58), pUC9:HPV59 (HPV59) and PBR322:HPV61 (HPV61).
  • oligonucleotides used as probes from Example 8 had chemical labels covalently attched at the ends distal from ligation. These labels were: 5'-fluorescein-LCRSA, 3'-fluorescein-LCRSA', 3'- biotin-LCR5B and 5'-biotin-LCR5B'. Covalent attachment was performed by known methods, i.e., reaction of amine-terminated oligonucleotides with FITC or biotin-NHS-ester essentially following the procedures of Kansal et al., Tet. Letters 29:5537-5540 (1988). The thermal cycler used was obtained from Coy Laboratory Products, Ann Arbor, MI.
  • the matrix was washed with buffer (0.3M Nacl, 10 mM TRIS pH8, 0,1%NaN 3 ), Any immune complexes attached to the glass matrix was detected by using alkaline phosphatase-labeled conjugate that catalyzed the hydrolysis of 4-methylumbelliferone.
  • the rate at which the 4-methylumbelliferone was generated on the matrix was proportional to the concentration of LCR product formed in the reaction mixture.
  • the graph shows the results obtained from performing LCR on 10 7 molecules of the targets in shown.
  • the rate shown is the rate of generation of 4-methylumbelliferone, and is expresssed as fluorescence counts/second/second.
  • Background signal is approximately 10 c/s/s, as shown by the amplification of human placental DNA.
  • the only values above background are those for sample containing HPV16, and those values are about 60 times background signal.
  • Plasmids which contained full-length papilloma virus inserts in pGEM3 were used as targets.
  • the plasmids used were those described in Example 9 .
  • All of the oligonucleotides used as probes obtained from Example 10 had chemical labels covalently attached at the ends distal from ligation.
  • the thermal cycler was obtained from Coy Laboratory Products, Ann Arbor, MI.
  • the graph dislays the results obtained from performing LCR on 10 7 molecules of the targets.
  • the rate shown is the rate of generation of 4-methylumbelliferone, and is expressed as fluorescence counts/second/second.
  • Background signal is approximately 15 c/s/s, as shown by the amplification of human placental DNA.
  • the only values above background are those for sample containing HPV 8, and those values are about 40 times background signal.
  • Plasmids which contained full-length papilloma virus inserts in pGEM3 were used as targets.
  • the plasmids were those of Example 9 All of the oligonucleotides from Example 12 which were used as probes had chemical labels covalently attached at the ends distal from ligation.
  • the thermal cycler was as described in Example 11.
  • Example 9 Following the LCR procedure of Examples 4 and 5, the mixtures were analyzed as described in Example 9 using the prototype version of the IMx instrument (Abbott Laboratories, Abbott Park, IL).
  • the graph shows the results obtained from performing LCR on 10 7 molecules of the targets.
  • the rate shown is the rate of generation of 4-methylumbelliferone, and is expressed as fluorescence counts/second/second.
  • Background signal is approximately 15 c/s/s, as shown by the amplification of human placental DNA.
  • the only values above background are those for sample containing HPV 18, and those values are about 80 times background signal.
  • Plasmids which contained full-length papilloma virus inserts in pGEM3 were used as targets. All of the oligonucleotides from Example 14 used as probes had chemical labels covalently attached at the ends distal from ligation. The thermal cycler was as described in Example 11.
  • the graph details the results obtained from performing LCR on 10 7 molecules of the targets.
  • the rate shown is the rate of generation of 4-methylumbelliferone, and is expressed as fluorescence counts/second/second.
  • Background signal is approximately 10 c/s/s, as shown by the amplification of human placental DNA.
  • the only values above background are those for sample containing HPV 16, and those values are about 36 times background signal.
  • the appendix lists the sequences of HPV types 6, 11, 16, 18, 31, and 33. It also shows where the sequences of this invention line up with respect to these HPV sequences. In addition, the appendix shows where other sequences, known to the Inventors as of 28 September 1990, and claimed or disclosed by or unknown to others, line up with respect to these sequences.

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Claims (17)

  1. Composition utile dans la LCR pour amplifier l'ADN de virus du papillome humain présent dans échantillon à doser, ladite composition comprenant un ensemble de quatre sondes oligonucléotidiques, lesdits ensembles de sondes étant sélectionnés dans le groupe constitué par les ensembles d'oligonucléotides suivants :
    Figure imgb0098
    Figure imgb0099
    Figure imgb0100
    Figure imgb0101
  2. Composition selon la revendication 1, destinée à amplifier l'ADN de virus du papillome humain de type 16 présent dans un échantillon à doser, ladite composition comprenant un ensemble de quatre sondes oligonucléotidiques, lesdits ensembles de sondes étant sélectionnés dans le groupe constitué par les ensembles d'oligonucléotides suivants :
    LCR5 (n° d'identification 81, 82, 83 et 84) et LCR8 (n° d'identification 93, 94, 95 et 96).
  3. Composition selon la revendication 1, destinée à amplifier l'ADN de virus du papillome humain de type 18 présent dans un échantillon à doser, ladite composition comprenant un ensemble de quatre sondes oligonucléotidiques, lesdits ensembles de sondes étant sélectionnés dans le groupe constitué par les ensembles d'oligonucléotides suivants :
    LCR6 (n° d'identification 85, 86, 87 et 88) et LCR7 (n° d'identification 89, 90, 91 et 92).
  4. Kit de détection de la présence d'ADN de virus du papillome humain dans un échantillon à doser, comprenant :
       une composition selon l'une quelconque des revendications 1 à 3, et en outre une ligase.
  5. Kit selon la revendication 4, dans lequel ladite ligase est thermostable.
  6. Composition utile dans la PCR pour amplifier l'ADN de virus du papillome humain présent dans un échantillon à doser, ladite composition comprenant :
    une première amorce d'acide nucléique de direction sens, capable de s'hybrider au brin antisens de l'ADN de HPV, ladite amorce ayant de 10 à environ 30 nucléotides de long et une séquence sélectionnée dans le groupe constitué par les séquences suivantes :
    Figure imgb0102
    une deuxième amorce d'acide nucléique de direction antisens, capable de s'hybrider au brin sens de l'ADN de HPV, ladite amorce ayant de 10 à environ 30 nucléotides de long et une séquence sélectionnée dans le groupe constitué par les séquences suivantes :
    Figure imgb0103
    pour autant que lesdites première et deuxième amorces s'hybrident à leurs brins respectifs antisens et sens à des emplacements tels que leurs extrémités 3' ne se chevauchent pas et que, dans la direction d'extension, les extrémités 5' desdites amorces soient plus espacées que les extrémités 3' desdites amorces.
  7. Composition selon la revendication 6, dans laquelle lesdites première et deuxième amorces sont sélectionnées parmi les paires suivantes de séquences oligonucléotidiques (identifiées par leur numéro d'identification) :
    1 et 5, 6 et 5, 7 et 5, 81 et 84,
    85 et 88, 89 et 92, et 93 et 96.
  8. Kit de détection de la présence d'ADN de virus du papillome humain dans un échantillon à doser, comprenant :
       une composition selon la revendication 6 ou 7 et
       en outre une polymérase.
  9. Kit selon la revendication 8, dans lequel ladite polymérase est thermostable.
  10. Procédé de détermination de la présence d'un virus quelconque du papillome humain dans un échantillon à doser, comprenant :
    a. l'hybridation de l'ADN dans l'échantillon à doser avec au moins un oligonucléotide consensus sélectionné dans le groupe de séquences constitué par :
    Figure imgb0104
    et leurs compléments, ledit oligonucléotide étant conjugué à un composé émetteur d'un signal, capable de produire un signal détectable, et
    b. la détermination de la présence du virus du papillome humain par détection du signal émis.
  11. Procédé de détermination de la présence du virus du papillome humain de type 16 dans un échantillon à doser, comprenant :
    a. l'hybridation de l'ADN dans l'échantillon à doser avec au moins un oligonucléotide sélectionné dans le groupe de séquences constitué par :
    Figure imgb0105
    et leurs compléments,
    ledit oligonucléotide étant conjugué à un composé émetteur d'un signal, capable de produire un signal détectable, et
    b. la détermination de la présence du virus du papillome humain par détection du signal émis.
  12. Procédé de détermination de la présence du virus du papillome humain de type 18 dans un échantillon à doser, comprenant :
    a. l'hybridation de l'ADN dans l'échantillon à doser avec au moins un oligonucléotide sélectionné dans le groupe de séquences constitué par :
    Figure imgb0106
    et leurs compléments,
    ledit oligonucléotide étant conjugué à un composé émetteur d'un signal, capable de produire un signal détectable, et
    b. la détermination de la présence du virus du papillome humain par détection du signal émis.
  13. Procédé selon une quelconque des revendications 10 à 12, comprenant en outre une étape d'amplification avant ou pendant ladite étape d'hybridation.
  14. Procédé selon la revendication 13, dans lequel ladite étape d'amplification comprend la PCR ou la LCR.
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US5342930A (en) * 1988-05-13 1994-08-30 Institut Pasteur Isolated DNA of human papillomavirus type 54(HPV54)
WO1995030009A2 (fr) * 1994-04-29 1995-11-09 Gatsby Charitable Foundation Identification, production et utilisation de saponine glycosyle hydrolases
US5554538A (en) * 1986-03-21 1996-09-10 Institut Pasteur DNA sequences derived from papillomavirus HPV-33 genome
EP0774518A2 (fr) 1995-11-15 1997-05-21 Gen-Probe Incorporated Sondes d'acides nucléiques complémentaires aux acides nucléiques du virus du Papillome humain, procédés associés et trousse d'essais
US5712092A (en) * 1984-11-30 1998-01-27 Institut Pasteur Papillomavirus probe and process for in vitro diagnosis of papillomavirus infections
US5876922A (en) * 1985-07-31 1999-03-02 Institute Pasteur Papillomavirus probe and a process for in vitro diagnosis of papillomavirus infections
US5888731A (en) * 1995-08-30 1999-03-30 Visible Genetics Inc. Method for identification of mutations using ligation of multiple oligonucleotide probes
US6352825B1 (en) * 1994-02-21 2002-03-05 Stichting Researchfonds Pathologie Human Papilloma Virus detection in a nucleic acid amplification process using general primers

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US6180338B1 (en) 1992-08-04 2001-01-30 Beckman Coulter, Inc. Method, reagent and kit for the detection and amplification of nucleic acid sequences
US6207368B1 (en) 1992-08-04 2001-03-27 Beckman Coulter, Inc. Methods and reagents for controlling chain extension and ligation chain reactions
EP0656070A4 (fr) * 1993-05-24 1996-03-20 Amoco Corp Sondes d'acides nucleiques destinees a des bacteries du genre legionella.
US7569344B2 (en) 1998-10-26 2009-08-04 Ventana Medical Systems, Inc. Detection of human papilloma virus in papanicolaou (Pap) smears
GB0018050D0 (en) * 2000-07-21 2000-09-13 Norchip As Detection of human papillomavirus mRNA
EP1302550A1 (fr) * 2001-10-10 2003-04-16 King Car Food Industrial Co., Ltd. Procédé et détecteur pour la détection de virus de type de HPV
JP2005537028A (ja) 2002-06-26 2005-12-08 ザ　ペン　ステート　リサーチ　ファウンデーション ヒト乳頭腫ウイルス感染症を治療する方法及び材料
PL1844164T3 (pl) 2005-11-15 2010-12-31 Genoid Kft Sposób wykrywania patogenów z zastosowaniem sond typu "latarni molekularnych"
WO2008089519A1 (fr) * 2007-01-25 2008-07-31 Symbion Pathology Pty Limited Procédé de criblage de papillomavirus humain
US9090948B2 (en) 2008-09-30 2015-07-28 Abbott Molecular Inc. Primers and probes for detecting human papillomavirus and human beta globin sequences in test samples

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FR2629458B2 (fr) * 1987-07-31 1991-08-09 Ire Celltarg Sa Nouvelles sondes d'acides nucleiques specifiques de differents types de virus de papillome humain
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CA1323293C (fr) * 1987-12-11 1993-10-19 Keith C. Backman Essai utilisant la reorganisation d'une sonde a l'acide nucleique dependant d'une matrice
JPH03503605A (ja) * 1988-04-04 1991-08-15 オンカー　インコーポレーテッド ヒトパピローマウイルス分類法および該方法に使用する核酸プローブ
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DE69022246T2 (de) * 1989-12-01 1996-02-29 Amoco Corp Detektion von hpv-transkripts.
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Cited By (17)

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US6391539B1 (en) 1984-11-30 2002-05-21 Institut Pasteur Immunogenic compositions of human papillomavirus
US5712092A (en) * 1984-11-30 1998-01-27 Institut Pasteur Papillomavirus probe and process for in vitro diagnosis of papillomavirus infections
US5876922A (en) * 1985-07-31 1999-03-02 Institute Pasteur Papillomavirus probe and a process for in vitro diagnosis of papillomavirus infections
US5554538A (en) * 1986-03-21 1996-09-10 Institut Pasteur DNA sequences derived from papillomavirus HPV-33 genome
US5648459A (en) * 1986-03-21 1997-07-15 Institut Pasteur Determined DNA sequences derived from a papillomavirus genome, their uses for in vitro diagnostic purposes and the production of antigenic compositions
US5876723A (en) * 1986-03-21 1999-03-02 Institut Pasteur Purified human papillomavirus type 33 (HPV-33) peptides as an immunogenic composition
US6344314B2 (en) 1986-03-21 2002-02-05 Institut Pasteur Determined DNA sequenced derived from a papillomavirus genome, their uses for in vitro diagnostic purposes and the production of antigenic compositions
US5342930A (en) * 1988-05-13 1994-08-30 Institut Pasteur Isolated DNA of human papillomavirus type 54(HPV54)
US5534439A (en) * 1988-05-13 1996-07-09 Institut Pasteur Isolated DNA of human papillomavirus type 55 (HPV55)
US5824466A (en) * 1988-05-13 1998-10-20 Institut Pasteur Purified polypeptide of human papillomavirus type 49 (HPV49) type 50 (HVP50), and type 55 (HPV55)
US6352825B1 (en) * 1994-02-21 2002-03-05 Stichting Researchfonds Pathologie Human Papilloma Virus detection in a nucleic acid amplification process using general primers
WO1995030009A2 (fr) * 1994-04-29 1995-11-09 Gatsby Charitable Foundation Identification, production et utilisation de saponine glycosyle hydrolases
US6025139A (en) * 1995-08-30 2000-02-15 Visible Genetics Inc. Method for identification of mutations using ligation of multiple oligonucleotide probes
US5888731A (en) * 1995-08-30 1999-03-30 Visible Genetics Inc. Method for identification of mutations using ligation of multiple oligonucleotide probes
AU726047B2 (en) * 1995-11-15 2000-10-26 Gen-Probe Incorporated Nucleic acid probes complementary to human papillomavirus nucleic acid and related methods and kits
WO1997018334A2 (fr) * 1995-11-15 1997-05-22 Gen-Probe Incorporated Sondes d'acides nucleiques complementaires des acides nucleiques du virus du papillome humain, methodes et preparations associees
EP0774518A2 (fr) 1995-11-15 1997-05-21 Gen-Probe Incorporated Sondes d'acides nucléiques complémentaires aux acides nucléiques du virus du Papillome humain, procédés associés et trousse d'essais

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JP2001231587A (ja) 2001-08-28
GR3021724T3 (en) 1997-02-28
DE69121821D1 (de) 1996-10-10
JP2003144182A (ja) 2003-05-20
KR920006510A (ko) 1992-04-27
JPH04281791A (ja) 1992-10-07
DK0477972T3 (fr) 1997-02-10
ES2094178T3 (es) 1997-01-16
DE69121821T2 (de) 1997-04-03
ATE142276T1 (de) 1996-09-15
AU8482691A (en) 1992-04-02
EP0477972A2 (fr) 1992-04-01

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